Large-scale production of proteins for biopharmaceutical applications involves the use of cell cultures that are known to produce proteins exhibiting varying levels of heterogeneity. One potential source of heterogeneity involves C-terminal lysine residues, such as those typically found on the heavy chains of antibody molecules. C terminal lysines can be lost, so that individual antibodies in a production batch can vary at their C terminus as to whether a lysine residue is present. C-terminal lysines can be potentially present on both the heavy chains of an antibody (Lys 2), on either one of the heavy chains (Lys 1), or neither of them (Lys 0). Since lysine can carry a positive charge, antibodies lacking the basic C-terminal lysine(s) differ in the charge state from the ones that contain the lysine, so that the distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchange chromatographic methods, such as analysis employing a ProPac WCX-10 Weak Cation-Exchange column for the high-resolution separation of protein isoforms (Dionex, CA), and subsequently quantified.
The C-terminal lysine heterogeneity is commonly observed in biopharmaceutical antibodies and proteins. For instance, in the process of manufacture of Remicade (Infliximab), the heterogeneity during the fermentation was approximately 20% (Lys 0 and Lys 1) and 80% (Lys 2) (US Patent Application publication US2010/0297697A1). Other examples are detailed in a review article on lysine variants (Harris R, “Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture” Journal of Chromatography A, 705 129-134 (1995)).
The present study is directed to cell culture methods to modulate a product quality attribute of recombinant proteins. Specifically, the invention provides methods for influencing the relative distribution of the different C-terminal lysine variants of the product antibody obtained from the cell culture harvest. C-terminal lysine can be potentially present on both the heavy chains of the antibody (Lys 2), on either one of them (Lys 1) or neither of them (Lys 0). Since, antibodies lacking the basic C-terminal lysine(s) differ in the charge state from the ones that contain the lysine, the distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the total Lysine Sum) can be detected by ion-exchange chromatographic methods such as WCX-10 and subsequently quantified (FIG. 1).